The invention relates to the polypeptides associated with the expression of a resistance to antibiotics of the glycopeptide family, this resistance being of a type inducible by vancomycin and not inducible by teicoplanin, particular in the Gram-positive bacteria, in particular in the family of the Gram-positive cocci. The invention also relates to a nucleotide sequence coding for these polypeptides. It also relates to the use of these polypeptides and their nucleotide sequence as agents for the in vitro detection of resistance to glycopeptides. Among the Gram-positive cocci, the invention relates more particularly to the enterococci, the streptococci and the staphylococci.
The glycopeptides, which include vancomycin and teicoplanin, are antibiotic inhibitors of the synthesis of the bacterial cell wall. These antibiotics are very much used for the treatment of severe infections due to Gram-positive coca (enterococci, streptococci and staphylococci) in particular in cases of allergy and resistance to the penicillins.
Up to 1986 vancomycin proved to be efficacious against almost all strains of enterococci.
The activity of the glycopeptides depends on the formation of a complex between the antibiotic and the peptidoglycan precursors more than on their direct interaction with enzymes of cell wall metabolism. In particular, it has been observed that the glycopeptides bind to the terminal D-alanyl-D-alanine (D-ala-D-ala) residues of the peptidoglycan precursors.
Several phenotypes of resistance to the glycopeptides have been demonstrated; in particular, strains resistant to a high level of glycopeptides and strains resistant to low concentration levels.
By strain resistant to a high level is meant a strain of bacteria, in particular a strain of Gram-positive cocci, for which the minimal inhibitory concentrations (MC) of vancomycin and teicoplanin are higher than 32 and 8 xcexcg/ml, respectively. The MIC of vancomycin towards strains with low-level resistance are included between 8 and 32 xcexcg/ml. The VanB phenotype is characterized by a resistance inducible by vancomycin but not inducible by teicoplanin. Once induced, this resistance may exist against different glycopeptides, in particular against vancomycin and/or teicoplanin, and at variable levels.
The strains of enterococci corresponding to the VanB phenotype (class B) are in particular strains of E. faecalis and E. faecium. 
Al-Obeici S et al. (FEMS Microbiology Letters 70 (1990) 101-106) have thus compared the resistance proteins to glycopeptides, inducible by vancanycin, in four strains at Enterococci, and have deduced from their comparison the existence of three types of proteins, one of these types being present in the E. faecium strain resistant to low levels of vancomycin. According to the authors of this publication, a protein of molecular weight of about 39.5 kDa is induced in the strains with low-level resistance and this resistance is linked to induction by vancomycin. These strains were also reported to exhibit a resistance to teicoplanin, also induced by vancomycin.
According to Al-Obeid et al., this protein of 39.5 kDa is present in multiple forms but the nature of this multiplicity has not been studied. According to these authors there might exist a structural specificity depending on the species of bacteria concerned and the level of resistance, which needs to be confirmed.
In this publication Al-Obeid et al. described 11 amino acids of the N-terminal sequence of the protein of 39.5 kDa and observed that this sequence exhibited about 70% homology with many membrane proteins of prokaryotic or eukaryotic origin having diverse functions. According to the authors this comparison did not allow the possible function of the protein to be established. Finally, Al-Obeid et al. noticed that other proteins are induced, although to a lesser degree.
The invention relates to peptides, polypeptides or proteins implicated in the expression of a resistance to antibiotics of the glycopeptide family and in particular to vancomycin and/or teicoplanin as well as nucleotide sequences coding for such polypeptides. The resistance in question above is of a type inducible by vancomycin but not by teicoplanin.
The expressions xe2x80x9cimplicated in the expression of a resistancexe2x80x9d or xe2x80x9cimplicated in a resistancexe2x80x9d signify that the protein of the invention is necessary in order for the resistance to be manifest.
The invention also relates to nucleotide probes utilizable for the detection of a resistance to the glycopeptides, in particular by means of the polymerase chain reaction (PCR), or by assays involving antibodies.
Thus, the object of the invention is a VanB protein characterized in that it comprises the following amino acid sequence I, and in that it participates in the resistance to glycopeptides, in particular to vancomycin, this resistance being of a type inducible by vancomycin and not by teicoplanin in Gram-positive bacteria (SEQ ID NO:2).
M N K I X V A I I F G G C S E E H D V S V K S A I E I A A N I N T E K F D P H Y I G I T K N G V W K L C K K P C T E W E A D S L P A I F S P D R K T H G L L V M K E R E Y E T R R I D V A F P V L H G K C G E D G A I Q G L F E L S G I P Y V G C D I Q S S A A C M D K S L A Y I L T K N A G I A V P E F Q M I E K G D K P E A R T L T Y P V F V K P A R S G S S F G V T K V N S T E E L N A A I E A A G Q Y D G K I L I E Q A I S G C E V G C A V M G N E D D L I V G E V D Q I R L S H G I F R I H Q E N E P E K G S E N A M I I V P A D I P V E E R N R V Q E T A K K V Y R V L G C R G L A R V D L F L Q E D G G I V L N E V N T L P G F T S Y S R Y P R M A A A A G I T L P A L I D S L I T L A I E R
By the expression xe2x80x9cinducible resistancexe2x80x9d is meant the capacity of a specific Gram-positive bacterium, in particular of a specific Enterococcus strain, to produce a VanB protein in the presence of a concentration of 0.05 to 1 xcexcl/ml of vancomycin.
The resistance to one or more defined glycopeptides may result in the persistence of an infection due to microbes usually sensitive to the glycopeptides, or may be detected by means of an antibiogram (particularly for high levels of resistance), the MIC, hybridization with probes (after amplification by the PCR, for example).
According to a first embodiment of the invention, the VanB protein is characterized in that it is implicated in an inducible resistance to glycopeptides, and in particular to vancomycin, in enterococci and for example in strains of the genus E. faecium or E. faecalis. 
The invention also relates to a VanB protein characterized in that it comprises an amino acid sequence modified with respect to sequence I by deletion, insertion, or replacement of one or more amino acids, provided that the VanB protein thus modified is implicated in Gram-positive bacteria in a resistance to glycopeptides, in particular to vancomycin, this resistance being of a type inducible by vancomycin, but not inducible by teicoplanin.
Also included in the framework of the invention is any peptide fragment of the VanB protein characterized in that it corresponds to the amino acid sequence I or any part of this sequence functionally associated with the inducible resistance to glycopeptides, in particular to vancomycin, in Gram-positive bacteria, for example bacteria of the family of the enterococci.
Advantageously peptide fragments of the invention exhibit additionally or alternatively antigenic properties and are hence recognized by antibodies formed against the VanB protein
A particular fragment of sequence I corresponds for example to the following sequence or includes this sequence (residues 110-305 of SEQ ID NO:2):
L F E L S G I P Y V G C D I Q S S A A C M D K S L A Y I L T K N A G I A V P E F Q M I E K G D K P E A R T L T Y P V F V K P A R S G S S F G V T K V N S T E E L N A A I E A A G Q Y D G K I L I E Q A I S G C E V G C A V M G N E D D L I V G E V D Q I R L S H G I F R I H Q E N E P E K G S E N A M I I V P A D I P V E E R N R V Q E T A K K V Y R V L G C R G L A R V D L F L Q E D G G I V L N E V
According to another embodiment of the invention, these antigens are specific for the VanB protein and thus not recognized by antibodies recognizing the VanA and VanC proteins such as described in the patent application EF 91920753.
In addition the invention relates to a nucleotide sequence characterized in that it codes for a VanB protein implicated in resistance to glycopeptides, in particular to vancomycin, in Gram-positive bacteria, this resistance being of a type inducible by vancomycin but not inducible by teicoplanin, said VanB protein comprising the amino acid sequence I, or in that it is a DNA sequence complementary to this coding sequence, or a corresponding RNA sequence.
By complementary sequence is meant any DNA sequence whose nucleotides are complementary to those of sequence I and whose orientation is reversed.
A particular nucleotide sequence corresponding to this definition is characterized in that it comprises the following nucleotide sequence II or a nucleotide sequence modified with respect to II provided that it codes for a protein implicated in resistance to glycopeptides, in particular to vancomycin, in Gram-positive bacteria, this resistance being of a type inducible by vancomycin but not inducible by teicoplanin (SEQ ID NO:1).
GAGCGTGTGCTGCGAGATACCACAGAAAACAATCAGAATTGTCTTAACTGAAA GGAGTTTACAGCATGAATAAAATAAAAGTCGCAATTATCTTCGGCGGTTGCTCGG AGGAACATGATGTGTCGGTAAAATCCGCAATAGAAATTGCTGCGAACATTAATAC TGAAAAATTCGATCCGCACTACATCGGAATTACAAAAAACGGCGTATGGAAGCTA TGCAAGAAGCCATGTACGGAATGGGAAGCCGATAGTCTCCCCGCCATATTCTCCC CGGATAGGAAAACGCATGGTCTGCTTGTCATGAAAGAAAGAGAATACGAAACTCG GCGTATTGACGTGGCTTTCCCGGTTTTGCATGGCAAATGCGGGGAGGATGGTGCG ATACAGGGTCTGTTTGAATTGTCTGGTATCCCCTATGTAGGCTGCGATATTCAAA GCTCCGCAGCTTGCATGGACAAATCACTGGCCTACATTCTTACAAAAAATGCGGG CATCGCCGTCCCCGAATTTCAAATGATTGAAAAAGGTGACAAACCGGAGGCGAGG ACGCTTACCTACCCTGTCTTTGTGAAGCCGGCACGGTCAGGTTCGTCCTTTGGCG TAACCAAAGTAAACAGTACGGAAGAACTAAACGCTGCGATAGAAGCAGCAGGACA ATATGATGGAAAAATCTTAATTGAGCAAGCGATTTCGGGCTGTGAGGTCGGCTGC GCGGTCATGGGAAACGAGGATGATTTGATTGTCGGCGAAGTGGATCAAATCCGGT TGAGCCACGGTATCTTCCGCATCCATCAGGAAAACGAGCCGGAAAAAGGCTCAGA GAATGCGATGATTATCGTTCCAGCAGACATTCCGGTCGAGGAACGAAATCGGGTG CAAGAAACGGCAAAGAAAGTATATCGGGTGCTTGGATGCAGAGGGCTTGCTCGTG TTGATCTTTTRSTGCAGGAGGATGGCGGCATCGTTCTAAACGAGGTCCAATACCC TGCCCGGTTTTACATCGTACAGCCGCTATCCACGCATGGCGGCTGCCGCAGGAAT CACGCTTCCCGCACTAATTGACAGCCTGATTACATTGGCGATAGAGAGGTGACCC GTATGGAAAATGGTTTTTTGTTTTTTAGATGAAATGTTGCA
Generally speaking, the object of the invention is also a nucleotide fragment characterized in that it is capable of hybridizing under stringent conditions with a sequence such as defined in sequent II above.
The stringent conditions are the following:
reaction temperature of 65xc2x0 C. overnight in a solution containing 0.1% SDS, 0.7% skimmed milk powder, 6xc3x97SSC (1xc3x97SSC=0.15M NaCl and 0.015M sodium citrate at pH=7.0)
washes at room temperature in 2=SSC-0.1% SDS, then at 65xc2x0 C. in 0.2 SSC-0.1% SDS.
Advantageously a nucleotide fragment corresponding to the previous definition will have at least 15 nucleotides, and preferably at least 20.
For this purpose a particular nucleotide sequence comprises the following sequence (SEQ ID NO:3)
TCTGTTTGAATTGTCTGGTATCCCCTATGTAGGCTGCGATATTCAAA GCTCCCCAGCTTGCATGGACAAATCACTGGCCTACATTCTTACAAAAAATGCGGG CATCGCCGTCCCCGAATTTCAAATGATTGAAAAAGCTGACAAACCGGAGGCGAGG ACGCTTACCTACCCTGTCTTTGTGAAGCCGGCACGGTCAGGTTCGTCCTTTGGCG TAACCAAAGTAAACAGTACGGAAGAACTAAACGCTGCGATAGAAGCACCAGGACA ATATGATGGAAAAATCTTAATTGAGCAAGCGATTTCGGGCTGTGAGGTCGGCTGC GCGGTCATGGGAAACGAGGATGATTTGATTGTCGGCGAAGTGGATCAAATCCGGT TGAGCCACGGTATCTTCCGCATCCATCAGGAAAACGAGCCGGAAAAAGGCTCAGA GAATGCGATGATTATCGTTCCAGCAGACATTCCGGTCGAGGAACGAAATCGGGTG CAAGAAACGGCAAAGAAAGTATATCGGGTGCTTGGATGCAGAGGGCTTGCTCGTG TTGATCTTTTTTTGCAGGAGGATGGCGGCATCGTTCTAAACGAGGTC
The peptides and polypeptides of the invention make it possible to define a genotypic class, characterized by the capacity of the nucleotide sequences coding for these peptides to hybridize under stringent conditions with the sequance II constituting a probe.
These fragments may be used as primers for carrying out amplification reactions, or as probes.
Particularly valuable probes correspond to the following sequences (SEQ ID NO:4-5).
primer 1: 5xe2x80x2 ATGGGAAGCCCATACTC 3xe2x80x2,(positions 241-258 of nucleotides of sequence I)
primer 2: 5xe2x80x2 GATTTCGTTCCTCGACC 3xe2x80x2 (complementary reverse sequence of the nucleotide fragment 860-877 of sequence I).
Nucleotide probes according to the invention may be specific for the detection in Gram-positive bacteria of sequences coding for a VanB protein implicated in the resistance to glycopeptides, in particular to vancomycin and/or teicoplanin, this resistance being inducible in conformity with the previous definition, these probes being in addition universal among these sequences.
By probes specific for VanB is meant any oligonucleotide hybridizing with a nucleotide sequence coding for a VanB protein according to the invention as described in the preceding pages, and not exhibiting cross-hybridization or amplification (PCR) reactions with sequences present in all of the sensitive strains.
A particular nucleotide fragment according to the invention is characterized in that it does not hybridize under stringent conditions with the DNA of strains of enterococci sensitive to vancomycin, in particular with the DNA of the strains E. faecalis JH2-2 and E. faecium BM4107.
These reference strains have bee described by Jacobs and Hobbs (J. Bacteria. 1a 1974, 360-372) and Leclercq et al. (Antimicrob. Agents Chemother. 33, 1989), respectively.
Another useful nucleotide fragment in the framework of the invention is specific for the vanB gene to the extent that it does not hybridize under stringent conditions with the vanA and vanC genes as described in the PCT application 91920753.
A particularly useful nucleotide fragment in the framework of the invention is the fragment corresponding to sequence II.
This fragment is an internal fragment derived from the gene implicated in the resistance to strains of enterococci. The resistance may exist at variable concentration levels of glycopeptides The invention also relates to nucleotide fragments modified with respect to the foregoing by mutation, addition or deletion of nucleotides, provided that the fragment thus modified either codes for a fragment of the functional VanB protein as regards its property of the resistance to glycopeptides, in particular to vancomycin, under conditions described above, or hybridizes with the vanB gene.
Should the nucleotide fragments be used as probes, labelling is performed by the standard techniques, As examples, radioactive or enzymatic markers should be used.
Nucleotide fragments according to the invention may be used as primers to carry out the amplification of the nucleic acid contained in a given biological sample, for example by PCR.
Moreover, the invention relates to a recombinant DNA sequence characterized in that it comprises a nucleotide sequence described above under the control of regulatory elements likely to be involved in the cloning and expression of a gene implicated in a resistance, of a type inducible by vancomycin and not inducible by teicoplanin, to antibiotics of the glycopeptide family, in particular vancomycin, in a defined host.
This gene implicated in the resistance is for example the vanB gene which comprises the nucleotide sequence II or any functional part in terms of inducible resistance derived from a sequence hybridizing with sequence II.
The invention also relates to a recombinant vector for the cloning and expression, characterized in that it comprises a nucleotide sequence described above at a site inessential for its replication, optionally under the control of regulatory elements likely to be involved in the expression of a resistance, of a type inducible by vancomycin and not by teicoplanin, to antibiotics of the glycopeptide family, in particular vancomycin, in a defined host.
Particular vectors are for example plasmids, phages, cosmids, YACs.
A preferred vector is the plasmid pAT201 deposited with the C.N.C.M on Dec. 11, 1992 under the number I-1277.
Another preferred vector is the plasmid pAT202 formed from the plasmid pUC19xcexa9 containing a 3.3 kb fragment containing the vanB gene of Enterococcus faecalis V583 (HindIII/KpnI).
pA202 was introduced into E. coli JM83 and deposited with the C.N.C.M on Mar. 29, 1993 under the number I-1291 (identification E. coli BM2973).
These vectors may be used to transform or transfect cell hosts in order to clone or express the nucleotide sequences of the invention.
A recombinant cell host according to the invention is characterized in that it is modified by a nucleotide sequence or a vector described above.
The cell host is preferably modified by this sequence under conditions permitting the expression of a functional VanB protein as regards inducible resistance to glycopeptides.
The object of the invention is also a recombinant VanB protein such as obtained from a recombinant cell host according to the previous definition, the VanB protein obtained being characterized in that its peptide skeleton comprises the above amino acid sequence, and in that it is implicated in a resistance to glycopeptides, in particular to vancomycin, in Gram-positive bacteria, this resistance being of a type inducible by vancomycin but not inducible by teicoplanin.
The VanB protein according to the invention makes it possible to prepare monoclonal or polyclonal antibodies characterized in that they recognize specifically the VanB protein or a peptide fragment described above.
These antibodies may be obtained according to the standard methods for the production of antibodies. In particular for the preparation of the monoclonal antibodies recourse should be had to the method of Kxc3x6hler and Milstein according to which monoclonal antibodies are prepared by cell fusion between myeloma cells and spleen cells of mice previously immunized with a polypeptide or a composition according to the invention, in conformity with the standard procedure.
The antibodies of the invention can advantageously be used for the detection of the presence at proteins characteristic of a resistance to the glycopeptides, in particular to vancomycin and teicoplanin, this resistance being of the type inducible by vancomycin but not inducible by teicoplanin.
Also included in the framework of the invention is a kit for the in vitro diagnosis in a biological sample of the presence of strains resistant to glycopeptides after induction, in particular by vancomycin but not by teicoplanin, these strains belonging in particular to the Gram-positive cocci, in particular in that they are strains of enterococci, for example E. faecium, characterized in that it contains:
optionally labelled antibodies described above,
a reagent for the detection of an immunological reaction of the antigen-antibody type,
optionally, reagents for lysing the cells of the tested sample,
optionally, a defined concentration of vancomycin to induce resistance.
The invention also relates to a kit such as that defined above which contains in addition antibodies specifically directed against the VanA protein and/or antibodies specifically directed against the VanC protein.
According to another embodiment of the invention, the kit enables resistance corresponding to a phenotype VanA, VanB or VanC to be detected indiscriminately and contains antibodies recognizing VanA, VanB and VanC. These antibodies may be selected by their capacity to recognize an epitope common to the three proteins. It may also be a mixture of antibodies recognizing different epitopes, specific to each of the proteins,
According to another embodiment at the invention, a kit for the in vitro diagnosis of the presence of strains resistant to low levels of glycopeptides, resistant in particular to vancomycin, is characterized in that it contains:
a nucleotide probe capable of hybridizing under stringent conditions with a nucleotide sequence of the vanB gene, and optionally,
nucleoside triphosphates dATP, dCTP, dTTP, dGTP,
a DNA polymerase.
Another detection kit contains in addition nucleotides capable of hybridizing specifically with the vanA gene and a probe capable of hybridizing specifically with the vanC gene.
This kit may be advantageously used for the detection of a resistance in Gram-positive cocci, in particular in enterococci, for example in E. faecium. 
The invention also relates to a kit for the in vitro detection of a resistance to glycopeptides, in particular to vancomycin, this resistance corresponding to one of the phenotypes VanA, VanB or VanC, the kit containing:
a nucleotide probe hybridizing with the genes vanA vanB and vanC,
nucleoside triphosphates dATP, dCTP, dTFP and dGTP,
a DNA polymerase.
The invention also relates to a procedure for the in vitro detection of the presence of strains resistant to glycopeptides, in particular to vancomycin and/or teicoplanin, these strains belonging in particular to the family of the Gram-positive cocci, in particular in that they are strains of enterococci, for example E. faecium or E. faecalis, characterized in that it comprises:
a) the placing of a biological sample likely to contain the resistant strains in contact with a primer constituted by a nucleotide fragment according to the invention such as that described above, capable of hybridizing with the nucleotide sequence under investigation and implicated in the expression at the resistance, this sequence being used as matrix in the presence of the 4 different nucleoside phosphates and a polymerase under conditions of hybridization such that for each nucleotide sequence having hybridized with a primer, an elongation product of each primer complementary to the matrix is synthesized,
b) the separation of the matrix from the elongation product obtained, this latter being then also able to behave as a matrix,
c) repetition of step a) so as to obtain a detectable quantity of the nucleotide sequences investigated,
d) the detection of the amplification product of the nucleotide sequences.
The probe used may thus be specific for the nucleotide sequence II or a sequence hybridizing with sequence II under stringent conditions. Under these conditions, the procedure according to the invention makes possible the detection of a resistance to glycopeptides, this resistance being inducible by vancomycin but not inducible by teicoplanin.
According to a particular embodiment of the invention, this procedure also comprises the placing of the biological sample in contact with a specific nucleotide fragment of the vanA gene and/or a specific nucleotide fragment of the vanC gene In this case the procedure according to the invention advantageously makes possible the detection of different phenotypes of resistance.
According to another embodiment, a resistance corresponding to a phenotype VanA, VanB or VanC will de detected indiscriminately by using a probe common to the genes vanA, vanB or vanC. Such a probe may be constructed from the aligned polypeptide sequences of FIG. 2.